ALBERT

Description: Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA–derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome...

Description: Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

Description: Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)

Description: Background: Platelets are critical for maintaining hemostasis, but inappropriate platelet activation can lead to pathogenic thrombosis. It has been demonstrated that the platelet integrin αIIbβ3 is essential for platelet aggregation and is also a major target antigen in immune thrombocytopenias (e.g. ITP). Current monoclonal antibodies (mAbs) against this protein complex have been generated using traditional methods involving cross-species immunization (e.g. mouse proteins into rat hosts). These approaches may generate a limited repertoire of anti-β3 mAbs since the antigenicity of the protein and the variety of epitopes targeted are based on amino acid sequence differences between the two species and integrin family members are highly conserved. Additionally, studies in murine models of ITP are hampered by the use of xenogeneic antibodies rather than syngeneic antibodies. Methods: We developed a method to generate mouse anti-mouse β3 integrin mAbs utilising β3 gene deficient mice (β3−/−) immunized with wild-type platelets. To generate antibodies specific to the PSI domain (HPA-1 region) of β3 integrin, β3−/− mice were immunized with the recombinant murine PSI domain of β3 integrin. Platelet binding and specificity were determined by flow cytometry and western blot. In vitro effects on platelet function were measured using aggregometry. Different doses of mAbs (5, 10, and 15 μg/mouse) were injected intravenously to induce thrombocytopenia in vivo. Results: A total of twelve mAbs were generated against native β3 integrin (JAN A1, B1, C1, D1 and DEC A1 and B1, 9D2, M1) or recombinant PSI domain (PSI A1, B1, C1, E1). The mAbs were specific for β3 integrin; no binding was observed using β3−/− platelets. Isotyping showed that DEC A1 and DEC B1 are IgG3, PSI E1 is IgG2b, and all other mAbs are IgG1. The anti-PSI domain mAbs recognized linear epitopes and the anti-native β3 mAbs recognized conformational epitopes. All mAbs, with the exception of JAN A1 and B1, cross-reacted with human platelets. JAN C1, JAN D1, DEC A1, 9D2, M1, and all anti-PSI antibodies inhibited mouse platelet aggregation. These antibodies, except DEC A1, 9D2 and M1, also inhibited human platelet aggregation. One anti-PSI domain antibody (PSI B1), however, directly induced human platelet aggregation in the absence of agonist in platelet rich plasma but not in PIPES buffer. This suggests that PSI B1 may initiate conformational changes in β3 integrin and promote fibrinogen binding. Six anti-β3 mAbs (JAN A1, B1, C1 and D1, 9D2 and M1) induced severe dose-dependent thrombocytopenia in mice, while the anti-PSI domain mAbs induced only a mild decrease in platelet count. Interestingly, the two IgG3 mAbs (DEC A1 and B1) did not induce thrombocytopenia. Conclusion: This approach to generating mouse anti-mouse β3 integrin mAbs using β3−/− mice was successful. Different anti-β3 mAbs had different effects on platelet aggregation, and on the induction of thrombocytopenia. These mAbs may be useful reagents for research in thrombosis and immune thrombocytopenia and as novel anti-thrombotic therapeutics.

Description: Abstract 3197 Background: Thrombosis and cardiovascular diseases (CVDs) result from blood vessel occlusion by inappropriately activated platelets. They are the leading causes of morbidity and mortality worldwide. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies have demonstrated that anthocyanins are antioxidative and prevent inflammation (J Biol Chem. 2005; 280:36792-01; Arterioscler Thromb Vasc Biol. 2007; 27:519-24), which may indirectly affect platelet function. It has also been reported that anthocyanins affect platelet activities in whole blood and platelet rich plasma (PRP). However, the direct effects of anthocyanins on platelet function and thrombus formation have not been studied. Methods: Here we investigated the effects of anthocyanins on thrombosis using purified platelets as well as several thrombosis models in vitro and in vivo. Cyaniding-3-gulucoside (Cy-3-g) and delphinidin-3-glucoside (Dp-3-g), the two predominantly bioactive compounds of anthocyanin preparations, were prepared from Polyphenol AS Company in Norway. Purified gel-filtered platelets and PRP from healthy human volunteers and C57BL/6J mice were incubated at 37°C for 10 minutes with different concentrations (0.5μM, 5μM and 50μM) of Cy-3-g, Dp-3-g or PBS buffer as a control. Platelet aggregation was assessed by aggregometry using 5μM ADP, 10μg/ml collagen, or 100μM thrombin receptor activating peptide (TRAP; AYPGKF) as agonists. Platelet adhesion and aggregation were assessed in response to an immobilized collagen matrix in an ex vivo perfusion chamber at both high (1800 s-1) and low (600 s-1) shear rates. The expression of activated GPIIbIIIa was determined via PAC-1 monoclonal antibody in flow cytometry. Lastly, the effects of anthocyanins on thrombus formation in C57BL/6J mice were assessed using a FeCl3-induced intravital microscopy thrombosis model. Results: Both Cy-3-g and Dp-3-g significantly inhibited platelet aggregation induced by collagen and TRAP in gel-filtered platelets, and inhibited aggregation induced by ADP, TRAP and collagen in human and mouse PRP. These inhibitory functions were observed at Cy-3-g and Dp-3-g doses as low as 0.5μM. Cy-3-g and Dp-3-g also reduced the surface expression of activated GPIIbIIIa on resting human platelets in a dose-dependent manner. These compounds also markedly reduced platelet adhesion and aggregation in perfusion chamber assays at both low and high shear rates. Using intravital microscopy, we further demonstrated that Cy-3-g and Dp-3-g decreased platelet deposition, destabilized thrombi, and prolonged the time required for thrombus formation and vessel occlusion. Conclusions: our data clearly demonstrated for the first time that anthocyanin compounds directly inhibited platelet activation, adhesion and aggregation, as well as attenuated thrombus growth at both arterial and veinous shear stresses. These effects on platelets likely contribute to the protective effects of anthocyanins against thrombosis and CVDs. Disclosures: No relevant conflicts of interest to declare.

Description: We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre+, Fnflox/flox, Fg/VWF−/−) mice and littermate control (Cre−, Fnflox/flox, Fg/VWF−/−) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti–mouse β3 integrin antibodies but was not affected by anti–mouse GPIbα antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl3-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Description: Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (〉98%) and platelet pFn (〉80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P

Description: Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (

Description: Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown. A single-molecule technique, biomembrane force probe (BFP), was employed to detect direct interactions between apoA-IV and platelet αIIbβ3 integrin. The BFP adhesion frequency assay demonstrated that apoA-IV bound to αIIbβ3 integrin on ADP treated platelets. ApoA-IV also bound to purified activated αIIbβ3 integrin or the integrin expressed on Chinese hamster ovary (CHO) cells. In comparison, apoA-IV did not significantly bind to αIIbβ3 integrin on resting platelets, GPIb-complex expressed on CHO cells, αMβ2 integrin expressed on K562 cells, nor purified α5β1 and αvβ3 integrins. Importantly, apoA-IV-αIIbβ3 interactions in these experiments could be completely inhibited by a blocking monoclonal antibody (M1) against β3 integrin. These data clearly demonstrated the specificity of apoA-IV for αIIbβ3 integrin. Furthermore, 2D kinetics measurements revealed that the effective 2D affinity of apoA-IV-αIIbβ3 is 43% of that between fibrinogen and αIIbβ3. The BFP competition assay showed that apoA-IV competitively inhibited fibrinogen-αIIbβ3 interactions at its physiological concentration. Platelet functional studies in vitro showed that recombinant apoA-IV significantly inhibited both mouse and human platelet aggregation following stimulation with various agonists. Consistently, platelet aggregation in platelet rich plasma of apoA-IV deficient mice (apoA-IV-/-) was enhanced. Depletion of human plasma apoA-IV also enhanced ADP-induced human platelet aggregation. In ex vivo perfusion chambers, recombinant apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV in blood enhanced ex vivo thrombus growth under both low and high shear stresses. Using two in vivo intravital microscopy thrombosis models and a carotid artery thrombosis model, we demonstrated that FeCl3- and laser-induced thrombosis were enhanced in apoA-IV-/-mice, while transfusion of recombinant apoA-IV markedly attenuated this process. In addition, we found recombinant apoA-IV significantly decreased platelet P-selectin expression, and consistently more P-selectin expression was observed on ADP treated platelets from apoA-IV-/- mice, suggesting that apoA-IV occupancy may inhibit fibrinogen or other prothrombotic ligands mediated αIIbβ3 outside-in signaling. We further found that the N-terminus of apoA-IV plays a key role in its inhibitory function and the exposure of N-terminus is negatively regulated by its C-terminus. Furthermore, mutation of the two aspartic acid (D) residues at apoA-IV N-terminal 5 and 13 abolished its binding for αIIbβ3 integrin as demonstrated by BFP adhesion frequency assay, resulting in the loss of these inhibitory effects.These findings suggest that D5 and D13 of apoA-IV are the potential binding sites for αIIbβ3 integrin. Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a new link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases. These findings may also contribute to hemostasis, P-selectin mediated postprandial platelet activation and inflammation. Disclosures No relevant conflicts of interest to declare.